(a) Field of the Invention
The present invention relates to the assessment of ultraviolet radiation UV effects on sunscreen-treated and non-treated human skin using a more sensitive technique. In particular, the Invention relates to:
the production of human skin substitutes containing keratinocytes, melanocytes, fibroblasts and dermal extracellular matrix;
the protection of human skin surface with a sunscreen using standard quantity (2 ml/cm2);
the irradiation of these treated and non-treated skin substitutes using UVA, UVB and simulated sunlight ultraviolet sources;
the analyses of the sunscreen effects on non-irradiated cutaneous cells and extracellular matrix; and
the analyses of he harmful effects of these UV irradiations on skin structure and the photo-induced DNA damage before and after sunscreen protection.
(b) Description of Prior Art
A sunny day generally puts people in a very good mood. Indeed, all of us look forward to outdoor activities in summer. Outdoor activities mainly depend on warm and sunny weather conditions. The sun seems to be innocuous for the vast majority of the world""s population. In fact, the sun simply provides life-sustaining heat, light and energy. However, solar irradiation causes a large variety of harmful effects including photoaging and skin cancers (Preston D S et al., 1992, N. Engl. J. Med., 327:1649-1662).
The sun is the main source of UV radiation (Young A R, 1990, Semin. Dermatol., 9(1) :25-31). The broad spectrum and intensity of UV light from the sun are due to the high temperature on the earth""s surface and its size. The intensity of solar UV reaching the earth is certainly highly dangerous and would probably be lethal to the majority, if not all living organisms on the earth""s surface, without the shielding afforded by ozone layer. The UV solar spectrum is divided into three categories.
Ultraviolet-C (UVC) radiation ranges from 200 to 280 nm and is completely absorbed by the earth""s ozone layer. Ultraviolet-B (UVB) radiation ranges from 280 to 320 nm. A significant amount is absorbed by the ozone layer. This type of radiation causes most sunburn, redness and skin cancer. Ultraviolet-A (UVA) radiation ranges from 320 to 400 nm. As opposed to UVC and UVB, very little of this UVA radiation is absorbed by the ozone layer. The UVA dose reaching the earth is 100 to 1,000 times higher than UVB radiation (Frederick J E et al., 1989, Photochem. Photobiol., 51:443-450).
Skin is exposed to UV radiations from birth to death. Although, there are few beneficial effects of exposure to UV light, most of their effects are deleterious to human skin and eyes (Kligman A M, 1969, JAMA, 210:2377-2380). The harmful effects of sunlight exposure result in erythema, accelerated skin aging, pigmentation anomalies, actinic keratosis, immunosuppression, DNA damage and skin cancers (Preston D S et al., 1992, N. Engl. J. Med., 327:1649-1662; Garmyn M et al., 1995, Dermatology, 190:305-308; Jeevan A et al., 1993, The Lancet, 342:1159-1161). Basic and clinical literature have incriminated both UVA and UVB on these different skin pathologies. Efforts to increase scientific knowledge regarding the photobiologic effects of UV light on the skin are important and relevant to both prevention and treatment of many human skin diseases.
The increasing need for sun protection agents to be used by the general population around the world has become evident over the last decade. In a population that spends more recreational time in the sun, there is a growing awareness regarding the protection of these people against the harmful effects of UV. The use of sunscreen designed to protect them has increased dramatically in the past decades (Stern R S et al., 1986, Arch Dermatol., 122:537-545). The generally accepted parameter for evaluating the efficacy of sunscreen preparations against UV radiation is the sun protection factor (SPF) (Jarratt M et al., 1983, J. Am. Acad. Dermatol., 9:354-362). The endpoint for the determination of a sunscreen SPF is the appearance of erythema which is related to protection against sunburn. The erythemal phase is evaluated 24 hrs following the irradiation protocol and calculated using the below defined formula:   SPF  =      "AutoLeftMatch"                  Minimal        ⁢                  xe2x80x83                ⁢        erythema        ⁢                  xe2x80x83                ⁢        dose        ⁢                  xe2x80x83                ⁢                  (                      J            ⁢                          /                        ⁢                          cm              2                                )                ⁢                  xe2x80x83                ⁢        in        ⁢                  xe2x80x83                ⁢        sunscreen        ⁢                  xe2x80x83                ⁢        protected        ⁢                  xe2x80x83                ⁢        skin                                                                        Minimal                ⁢                                  xe2x80x83                                ⁢                erythema                ⁢                                  xe2x80x83                                ⁢                dose                ⁢                                  xe2x80x83                                ⁢                                  (                                      J                    ⁢                                          /                                        ⁢                                          cm                      2                                                        )                                            ⁢                              xe2x80x83                                                                                        in              ⁢                              xe2x80x83                            ⁢              non              ⁢                              -                            ⁢              sunscreen              ⁢                              xe2x80x83                            ⁢              protected              ⁢                              xe2x80x83                            ⁢              skin                                          
UVB radiations are most effective for producing tanning as well as erythema (Pathak M, 1985, Ann. NY Acad. Sci., 453:328-339) and SPF values reflect the degree of protection against this UVB radiation. Recently produced sunscreens cover a wider UV spectrum to protect skin from ultraviolet irradiation including UVA. The evaluation of a sunscreen efficacy is based on SPF method. However, for different reasons this method cannot be used successfully as an endpoint in determining the efficacy of a sunscreen for blocking the UVA harmful effects.
UVA necessitate prolonged exposure period to initiate erythema in normal skin. To use the erythemal reaction as an indication of effectiveness against UVA radiation and to expedite the determination, the skin must be sensitized by the application of a phototoxic chemical prior to exposure to the UVA radiation. Since many of these chemicals are carcinogenic, using this technique is presently prohibited for ethical reasons (Groves A G, 1990, In Sunscreens Development, evaluation, and regulatory aspects (eds) Nicholas J Lowe and Nadim A Shaath. Marcel Dekker, inc New York:411-420).
Other possible endpoints for evaluating UV protection and basically UVA protection include immediate and delayed pigmentation. Immediate pigment darkening (IPD) is an oxygen-dependent photochemical reaction of melanin or its precursors in existing melanosomes (Beitner H, 1988, Photodermatology, 5:96-100). The response is a gray-brown pigmentation which appears during or immediately after irradiation and fades over a period ranging from a few minutes to a few hours (Kaidbey K H et al., 1978, J. Invest. Dermatol., 72:253-256). Delayed pigmentation (tanning) represents proliferation of melanocytes and increased production of melanosomes.
However, to get a complete efficacy against UVA and UVB radiations, the sunscreen must protects also against photoaging, DNA damage and skin cancers (Lowe J N et al., 1980, J. Invest. Dermatol., 74:181-182; Freeman S E et al., 1988, Photodermatology, 5:243-247).
The sunscreen predictive actions are generated using human volunteers (Lowe J N et al., 1980, J. Invest. Dermatol., 74:181-182). Unfortunately, these evaluations were costly, difficult and suspicious upon the development of hyperpigmented areas subsequent to UV exposure (Bridges B et al., 1980, Nature, 283:523-524).
To overcome these limitations, experimental models (rats, mice, etc.) were used for photodermatological studies (Wolf P et al., 1993, J. Invest. Dermatol., 101:523-527). Despite many similarities, the use of animal skin to elucidate mechanisms of UV-induced damage in human cutaneous tissues is hampered by inherent biological differences. Consequently, scientists are still looking for the appropriate model to:
1xe2x80x94understand the different mechanisms involved in skin damage after UV radiation,
2xe2x80x94be used for sunscreen testing;
3xe2x80x94be used as a more sensitive and effective method for UV harmful effect assessment and sunscreen efficacy concerning skin cancer prevention.
Regarding the third point, as the erythemal phase is due to the penetration into the skin of an important UV radiation dose which induces an inflammatory reaction first, and then erythema. Our concern is that prior to the suberythemal phase, UV radiation has already caused histological perturbations to the skin and DNA damage to the cutaneous cells (keratinocytes, melanocytes, etc.) which precede skin cancer. Consequently, it is mandatory to develop a more sensitive method that allows UV damage assessment before getting to the erythemal phase.
One of our interests is to devise a more sensitive method for skin protection against harmful UVA and UVB effects. This critical goal may be attained with the invention described below.
The present invention relates to a process of producing in vitro skin substitutes containing human fibroblasts included into a collagen gel over-layered with human keratinocytes. After 20 days of culture, these skin substitutes were either treated or non-treated with a sunscreen (applied on the epidermal surface), then exposed or not to UVA, UVB and simulated sunlight (UVA-UVB). A novel aspect of the invention is the correlation between structural damage on the skin substitutes and the DNA damage in the cells after each type of irradiation. These assessing methods (structural and DNA damage methods) are applicable to UVA, UVB and simulated sunlight harmful effect analyses. Furthermore, due to its high sensitivity, this DNA damage method is also useful to assess the deleterious effects of other rays such as X-ray and infrared wavelengths. This method is also useful to assess the toxicity of different chemicals and other products. Using these methods we will be able to draft new legislation dealing with a cancer protective factors as a criterion for predicting the ability of a sunscreen to provide protection against ultraviolet radiations (UVA, UVB, UVC).
It is predicted that using DNA damage would help in the determination of the real damage induced to the cells and the capacity of these chemical and physical toxic exposed cells to repair, die or mis-repair without death (mutations) leading to skin cancer.
Skin substitutes are very useful for UV effect assessment on cutaneous cells and extracellular matrix. Indeed, skin substitutes can be produced at large. Their use provides more effective and repeated results. Finally, skin substitutes avoid the use of human volunteers to assess the efficacy of sunscreen. Accordingly, after skin substitute production and irradiation, the DNA damage analysis provides a very sensitive method to assess the capacity of the skin to support one or the other ultraviolet doses.
The DNA damage analysis method provides a more sensitive way to assess the capacity of a sunscreen on preventing DNA damage in UVA, UVB, UVC and simulated sunlight irradiated skin.
The DNA damage analysis method also provides a very sensitive way to assess the capacity of a sunscreen in preventing DNA damage in X-ray and infrared wavelength damaged skin.
In addition, the DNA damage analysis method provides a more sensitive way to assess the toxicity of chemical in the skin.